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Mouse Fbxl5 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Fbxl5 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Fbxl5 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Fbxl5 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Fbxl5 gene (NCBI Reference Sequence: NM_001159963 ; Ensembl: ENSMUSG00000039753 ) is located on Mouse chromosome 5. 11 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 11 (Transcript: ENSMUST00000047857). Exon 4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Fbxl5 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-430B21 as template. Cas9, gRNA and targeting vector will be co-injected into fertilized eggs for cKO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice homozygous for a null mutation display embryonic lethality before turning of the embryo with iron overload, growth retardation, and hemorrhage. Mice heterozygous for a knock-out allele exhibit abnormal iron homeostasis when fed a low iron diet. Exon 4 starts from about 19.18% of the coding region. The knockout of Exon 4 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 2486 bp, and the size of intron 4 for 3'-loxP site insertion: 2572 bp. The size of effective cKO region: ~687 bp. The cKO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 4 11 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Fbxl5 Homology arm cKO region loxP site Page 2 of 8 https://www.alphaknockout.com Overview of the Dot Plot Window size: 10 bp Forward Reverse Complement Sequence 12 Note: The sequence of homologous arms and cKO region is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis. Overview of the GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(7187bp) | A(29.89% 2148) | C(17.62% 1266) | T(33.07% 2377) | G(19.42% 1396) Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. No significant high GC-content region is found. So this region is suitable for PCR screening or sequencing analysis. Page 3 of 8 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN -------------------------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr5 - 43768501 43771500 3000 browser details YourSeq 251 978 2967 3000 93.8% chr2 + 72527737 72595272 67536 browser details YourSeq 192 525 1164 3000 79.7% chr8 - 25188070 25188381 312 browser details YourSeq 182 525 1165 3000 86.4% chr7 + 25189277 25189839 563 browser details YourSeq 181 525 1161 3000 80.2% chr15 + 83247654 83247945 292 browser details YourSeq 171 525 1168 3000 85.0% chr5 + 116051191 116051741 551 browser details YourSeq 168 552 1166 3000 87.7% chr9 - 45897306 45897879 574 browser details YourSeq 167 525 1161 3000 88.4% chr4 + 136641286 136641878 593 browser details YourSeq 165 987 1181 3000 94.6% chr17 - 35054658 35054887 230 browser details YourSeq 165 987 1165 3000 97.2% chr15 - 100408808 100408997 190 browser details YourSeq 163 984 1165 3000 95.1% chr8 - 80690227 80690411 185 browser details YourSeq 163 528 1164 3000 83.8% chr1 + 175911569 175911866 298 browser details YourSeq 161 988 1178 3000 90.4% chr16 + 56084552 56084737 186 browser details YourSeq 159 987 1164 3000 95.0% chr11 - 115668869 115669047 179 browser details YourSeq 158 987 1166 3000 94.5% chr3 - 138469356 138469540 185 browser details YourSeq 157 987 1164 3000 94.4% chrX - 36679987 36680180 194 browser details YourSeq 157 988 1169 3000 95.0% chr3 + 107851419 107851610 192 browser details YourSeq 157 957 1166 3000 87.4% chr1 + 190021585 190021786 202 browser details YourSeq 156 987 1167 3000 93.3% chr6 - 38325870 38326057 188 browser details YourSeq 156 525 1167 3000 82.7% chrX + 151102306 151102602 297 Note: The 3000 bp section upstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. BLAT Search Results (down) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr5 - 43764814 43767813 3000 browser details YourSeq 113 922 1577 3000 89.1% chr11 - 98899498 99109346 209849 browser details YourSeq 91 1506 1627 3000 91.1% chr5 + 106755689 106755840 152 browser details YourSeq 91 1506 1629 3000 89.7% chr11 + 97205633 97205761 129 browser details YourSeq 85 1516 1627 3000 93.0% chrX + 48647397 48647539 143 browser details YourSeq 81 890 1010 3000 89.3% chr9 + 102834182 102834314 133 browser details YourSeq 80 1507 1622 3000 87.0% chr9 + 4202476 4202616 141 browser details YourSeq 76 1528 1620 3000 91.4% chr8 + 127141391 127141510 120 browser details YourSeq 71 1529 1622 3000 90.9% chr2 + 21403876 21403992 117 browser details YourSeq 70 1521 1609 3000 92.6% chr18 - 24593008 24593117 110 browser details YourSeq 69 1541 1627 3000 90.9% chr2 - 27510276 27510398 123 browser details YourSeq 69 1498 1619 3000 82.8% chr1 - 191701237 191701346 110 browser details YourSeq 67 894 981 3000 86.8% chr11 + 76290169 76290255 87 browser details YourSeq 66 894 998 3000 80.0% chr10 + 5051978 5052079 102 browser details YourSeq 65 152 1627 3000 90.2% chr12 + 24257741 24515722 257982 browser details YourSeq 63 108 281 3000 93.3% chr14 - 78778650 78778827 178 browser details YourSeq 63 148 273 3000 87.1% chr2 + 71498299 71893764 395466 browser details YourSeq 62 149 279 3000 91.9% chr12 + 4268245 4268382 138 browser details YourSeq 61 1507 1577 3000 94.3% chr8 - 71211058 71211129 72 browser details YourSeq 61 1560 1629 3000 94.3% chr3 + 108984893 108984963 71 Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Fbxl5 F-box and leucine-rich repeat protein 5 [ Mus musculus (house mouse) ] Gene ID: 242960, updated on 12-Aug-2019 Gene summary Official Symbol Fbxl5 provided by MGI Official Full Name F-box and leucine-rich repeat protein 5 provided by MGI Primary source MGI:MGI:2152883 See related Ensembl:ENSMUSG00000039753 Gene type protein coding RefSeq status VALIDATED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Fbl4; Fir4 Expression Ubiquitous expression in cerebellum adult (RPKM 17.0), placenta adult (RPKM 16.2) and 28 other tissues See more Orthologs human all Genomic context Location: 5; 5 B3 See Fbxl5 in Genome Data Viewer Exon count: 11 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (43744618..43782149, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (44135857..44173388, complement) Chromosome 5 - NC_000071.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 12 transcripts Gene: Fbxl5 ENSMUSG00000039753 Description F-box and leucine-rich repeat protein 5 [Source:MGI Symbol;Acc:MGI:2152883] Gene Synonyms Fbl4, Fir4 Location Chromosome 5: 43,744,615-43,821,638 reverse strand. GRCm38:CM000998.2 About this gene This gene has 12 transcripts (splice variants), 227 orthologues, 17 paralogues, is a member of 1 Ensembl protein family and is associated with 17 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Fbxl5-202 ENSMUST00000087465.10 4237 623aa ENSMUSP00000084733.4 Protein coding CCDS19263 Q8C2S5 TSL:1 GENCODE basic Fbxl5-201 ENSMUST00000047857.15 2858 690aa ENSMUSP00000045792.9 Protein coding CCDS51489 Q8C2S5 TSL:1 GENCODE basic APPRIS P2 Fbxl5-211 ENSMUST00000196483.4 2814 689aa ENSMUSP00000143703.1 Protein coding - Q8C2S5 TSL:1 GENCODE basic APPRIS ALT1 Fbxl5-204 ENSMUST00000119523.7 2801 673aa ENSMUSP00000113557.1 Protein coding - D3Z584 TSL:5 GENCODE basic Fbxl5-203 ENSMUST00000114047.9 2762 684aa ENSMUSP00000109681.3 Protein coding - Q8C2S5 TSL:1 GENCODE basic Fbxl5-209 ENSMUST00000141902.7 2517 611aa ENSMUSP00000120338.1 Protein coding - F6W6I1 CDS 5' incomplete TSL:5 Fbxl5-207 ENSMUST00000124610.5 2306 679aa ENSMUSP00000116720.2 Protein coding - F7BZC4 TSL:5 GENCODE basic Fbxl5-205 ENSMUST00000121736.5 2144 580aa ENSMUSP00000112444.1 Protein coding - Q8C2S5 TSL:1 GENCODE basic Fbxl5-212 ENSMUST00000199055.1 374 107aa ENSMUSP00000142582.1 Protein coding - A0A0G2JE06 CDS 3' incomplete TSL:5 Fbxl5-206 ENSMUST00000124421.1 3833 No protein - Retained intron - - TSL:1 Fbxl5-210 ENSMUST00000143316.7 2731 No protein - Retained intron - - TSL:1 Fbxl5-208 ENSMUST00000140469.2 397 No protein - Retained intron - - TSL:2 Page 6 of 8 https://www.alphaknockout.com 97.02 kb Forward strand 43.74Mb 43.76Mb 43.78Mb 43.80Mb 43.82Mb Genes Cc2d2a-201 >protein coding Gm43182-201 >TEC Bst1-204 >nonsense mediated decay (Comprehensive set... Cc2d2a-202 >protein coding Bst1-202 >retained intron Bst1-201 >protein coding Contigs AC140277.3 > Genes (Comprehensive
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